FINAL REPORT

Study Title

MICRONUCLEUS TEST IN BONE MARROW CELLS OF THE RAT

WITH EXP1313100

Author

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s-Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 505691

Substance 205622/A

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Final Report
EXP1313100 Project 505691

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1. CONTENTS 

1. CONTENTS .................................................................................................................................. 2

2. STATEMENT OF GLP COMPLIANCE.......................................................................................... 3

3. QUALITY ASSURANCE STATEMENT ......................................................................................... 4

4. SUMMARY ................................................................................................................................... 6

5. INTRODUCTION .......................................................................................................................... 7

5.1. Preface ................................................................................................................................. 7

5.2. 7

5.3. Aims of the study................................................................................................................... 7

5.4. Guidelines ............................................................................................................................. 8

5.5. Storage and retention of records and materials ..................................................................... 8

6. MATERIALS AND METHODS ...................................................................................................... 8

6.1. Test substance ...................................................................................................................... 8

6.1.1. Test substance information ......................................................................................... 8

6.1.2. Study specific test substance information .................................................................... 8

6.1.3. Test substance preparation ......................................................................................... 9

6.2. Reference substances .......................................................................................................... 9

6.2.1. Negative control .......................................................................................................... 9

6.2.2. Positive control ............................................................................................................ 9

6.3. Test system........................................................................................................................... 9

6.4. Animal husbandry ................................................................................................................10

6.5. Treatment ............................................................................................................................10

6.6. Observations ........................................................................................................................11

6.7. Study design ........................................................................................................................11

6.7.1. Dose range finding study............................................................................................11

6.7.2. Micronucleus main test...............................................................................................11

6.7.3. Isolation of bone marrow ............................................................................................11

6.7.4. Preparation of bone marrow smears ..........................................................................11

6.7.5. Staining of the bone marrow smears ..........................................................................12

6.7.6. Analysis of the bone marrow smears for micronuclei ..................................................12

6.8. Electronic data capture.........................................................................................................12

6.9. Interpretation ........................................................................................................................12

6.9.1. Acceptability of the assay ...........................................................................................12

6.9.2. Data evaluation and statistical procedures .................................................................12

6.10. List of deviations ........................................................................................................13

6.10.1. List of protocol deviations ......................................................................................13

6.10.2. List of standard operating procedures deviations ...................................................13

7. RESULTS ....................................................................................................................................13

7.1. Dose range finding study......................................................................................................13

7.2. Micronucleus main test.........................................................................................................13

7.2.1. Mortality and toxic signs .............................................................................................13

7.2.2. Micronucleated polychromatic erythrocytes ................................................................13

7.2.3. Ratio polychromatic to normochromatic erythrocytes .................................................14

7.2.4. Chemical analysis of dose preparations .....................................................................14

8. CONCLUSION.............................................................................................................................14

9. REFERENCES ............................................................................................................................14

TABLES 

Table 1 Mean body weight immediately prior to dosing with EXP1313100 ..............................15

Table 2 Mean number of micronucleated polychromatic erythrocytes and ratio of 

polychromatic/normochromatic erythrocytes ..............................................................15

APPENDICES 

APPENDIX 1 TABLES .....................................................................................................................15

APPENDIX 2 INDIVIDUAL DATA ....................................................................................................16

APPENDIX 3 STATISTICS ..............................................................................................................17

APPENDIX 4 HISTORICAL CONTROL DATA FOR MICRONUCLEUS STUDIES...........................18

APPENDIX 5 CERTIFICATE OF ANALYSIS ...................................................................................19

APPENDIX 6 FORMULATION ANALYSIS .......................................................................................20

Final Report

EXP1313100 Project 505691

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3. QUALITY ASSURANCE STATEMENT 

WIL Research Europe B.V., ‘s-Hertogenbosch, The Netherlands 

This report was inspected by the WIL Research Europe Quality Assurance Unit to confirm that the 

methods and results accurately and completely reflect the raw data. 

During the on-site process inspections, procedures applicable to this type of study were inspected. 

The dates of Quality Assurance inspections are given below. 

Project 505691 

Type of 

Inspections Phase/Process 

Start Inspection 

date 

End Inspection 

date Reporting date 

Study Protocol 27-May-2014 27-May-2014 27-May-2014

Protocol Amendment 01 04-Jul-2014 04-Jul-2014 04-Jul-2014

Protocol Amendment 02 09-Jul-2014 09-Jul-2014 09-Jul-2014

Specimen processing 10-Jul-2014 10-Jul-2014 10-Jul-2014

Protocol Amendment 03 07-Aug-2014 07-Aug-2014 07-Aug-2014 

Protocol Amendment 04 03-Sep-2014 03-Sep-2014 03-Sep-2014

Protocol Amendment 05 08-Sep-2014 08-Sep-2014 08-Sep-2014

Report 24-Oct-2014 24-Oct-2014 24-Oct-2014

Protocol Amendment 06 30-Oct-2014 30-Oct-2014 30-Oct-2014

Process 

Genetic and In Vitro 

Toxicology 17-Jun-2014 30-Jun-2014 30-Jun-2014 

Test Substance Handling 

Exposure 

Observations/Measurements 

Specimen Handling 

Animal Facilities 07-Jul-2014 18-Jul-2014 18-Jul-2014

Test Substance Handling 

Exposure 

Observations/Measurements 

Specimen Handling 

Test Substance Receipt 24-Jul-2014 31-Jul-2014 01-Aug-2014 

Test Substance Handling 

Analytical and physical 

chemistry 05-Aug-2014 12-Aug-2014 13-Aug-2014 

Test Substance Handling 

Exposure 

Observations/Measurements 

Specimen Handling 

Necropsy 15-Sep-2014 26-Sep-2014 30-Sep-2014

Observations/Measurements 

Specimen Handling 

Final Report

EXP1313100 Project 505691

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4. SUMMARY 

Micronucleus test in bone marrow cells of the rat with EXP1313100. 

EXP1313100 was tested in the Micronucleus Test in rats, to evaluate its genotoxic effect in developing 

erythrocytes (polychromatic erythrocytes) in the bone marrow. 

The study procedures described in this report were based on the most recent OECD and EC

guidelines. 

Batch E00873-025-001 of EXP1313100 was a brown solid. The test substance was suspended in 

propylene glycol. 

The concentrations analysed in the formulations of Group B/C, Group D an Group E were in 

agreement with target concentrations (i.e. mean accuracies between 85 and 115%). 

A small response at the retention time of the test substance was observed in the chromatograms of 

the Group A formulations. It was considered not to derive from the formulation since a similar 

response was obtained in the analytical blanks. 

Analysis of Group B/C and Group E formulations after storage yielded a relative difference of ≤ 10%. 

Based on this, the formulations were found to be stable during storage at room temperature under 

normal laboratory light conditions for at least 4 hours. 

In the dose range finding study 3 males and 3 females were dosed via oral gavage with 2000 mg 

EXP1313100 per kg body weight. The animals showed no treatment related clinical signs or mortality 

after dosing. Since there were no substantial differences in toxicity between sexes only males were 

used in the main study. 

In the main study male animals were dosed by oral gavage with vehicle or with 2000, 1000 and 

500 mg EXP1313100 per kg body weight. A positive control group was dosed once by oral gavage 

with 20 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups were used, each 

consisting of 5 animals. No treatment related clinical signs or mortality were noted in any animal 

treated with EXP1313100 or control animals receiving vehicle or cyclophosphamide. 

Bone marrow of the groups treated with EXP1313100 was sampled 24 or 48 (highest dose only) hours 

after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours 

after dosing, respectively. 

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the 

bone marrow of animals treated with EXP1313100 compared to the vehicle treated animals. 

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control 

animals was within the historical solvent control data range. Cyclophosphamide, the positive control 

substance, induced a statistically significant increase in the number of micronucleated polychromatic 

erythrocytes. Hence, both criteria for an acceptable assay were met. 

The groups that were treated with EXP1313100 showed no decrease in the ratio of polychromatic to 

normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of 

toxic effects of this test substance on erythropoiesis. The group that was treated with 

cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic 

erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis. 

It is concluded that EXP1313100 is not clastogenic or aneugenic in the bone marrow micronucleus 

test when sampled at 24 and 48 hours post dosing of male rats up to a dose of 2000 mg/kg the 

recommended dose in accordance with current regulatory guidelines under the experimental 

conditions described in this report. 

Final Report